Application
Note: 403
Analysis of Chloroacetanilide and Other
Acetamide Herbicide Degradates in Drinking
Water by LC-MS/MS
Jonathan Beck, Thermo Fisher Scientific, San Jose, CA, USA
Key Words
• TSQ Quantum
• Pesticides
Introduction
LC Conditions
Chloroacetanilide herbicide compounds in drinking water,
specifically ethanesulfonic acid (ESA) and oxanillic acid
(OA) degradates, may be found in water supplies near
agricultural production areas and are used on various
crops including corn and soybeans. Therefore, monitoring
of these compounds is of interest to various regulatory
agencies around the world.
The degradates of chloroacetanilide herbicide compounds
are polar, making them ideal compounds for analysis by
negative electrospray ionization mass spectrometry. This
application note illustrates the direct analysis of drinking
water spiked with chloroacetanilide herbicide compounds.
Excellent linearity and reproducibility are demonstrated
over the entire calibration range.
The gradient used in this experiment is shown in Table 2.
Experimental Conditions
Samples
The following chloroacetanilide herbicide compounds
were purchased from Chem Service, Inc. (West Chester,
PA): Acetochlor ESA, Alachlor ESA, Dimethenamid ESA,
Flufenacet ESA, Metolachlor ESA, Propachlor ESA,
Propachlor OA, Butachlor ESA, and Dimethachlor ESA.
Stock solutions were prepared in methanol. Drinking
water, purchased commercially, was spiked with these
compounds prior to analysis and was used for all
subsequent dilutions from stock solutions. Butachlor ESA,
the internal standard for the analysis, was added to all
samples at a concentration of 116 ng/mL. Dimethachlor
ESA, the surrogate, was added to all samples at a level of
90 ng/mL. Calibrators were prepared over five levels, and
the values are shown in Table 1.
Compound
Acetochlor ESA
Alachlor ESA
Dimethenamid ESA
Flufenacet ESA
Metolachlor ESA
Propachlor ESA
Propachlor OA
Butachlor ESA ISTD
Dimethachlor ESA SURR
Cal 01
Cal 02
Cal 03
Cal 04
Cal 05
180
150
90
158
112
286
56
116
90
90
75
45
79
56
143
28
116
90
18
15
9
15.8
11.2
28.6
5.6
116
90
9
7.5
4.5
7.9
5.6
14.3
2.8
116
90
3.6
3
1.8
3.16
2.24
5.72
1.12
116
90
Table 1: Actual concentrations for all analytes in the analysis. All
concentrations are given in ng/mL (ppb).
Instrument:
Autosampler:
Column:
Column Temperature:
Flow Rate:
Injection Volume:
Mobile Phase A:
Mobile Phase B:
Thermo Scientific Accela pump
Accela™ autosampler
Thermo Scientific Hypersil GOLD 50 x 2.1 mm, 3 µm
65 °C
0.25 mL/min
25 µL – Full Loop Mode
5 mM Ammonium Acetate in Water
Methanol
Time
%A
%B
0.0
7.0
10.0
18.0
20.0
25.0
25.1
40.0
90
80
75
75
20
20
90
90
10
20
25
25
80
80
10
10
Table 2: Gradient program used
for the analysis. The flow rate
was 0.25 mL/min using a
Hypersil GOLD™ 50 x 2.1 mm
3 µm column heated to 65 °C.
MS Conditions
MS:
Source:
Ionization:
ESI Voltage:
Sheath Gas:
Auxiliary Gas:
Capillary Temp:
Thermo Scientific TSQ Quantum Discovery MAX
Heated-Electrospray (H-ESI)
Negative ESI
3500 V
30 units
15 units at 300 °C
300 °C
Precursor and product ions were optimized via direct
syringe infusion. The optimized conditions for each
compound are given in Table 3.
Compound
Acetochlor ESA
Alachlor ESA
Dimethenamid ESA
Flufenacet ESA
Metolachlor ESA
Propachlor ESA
Propachlor OA
Butachlor ESA ISTD
Dimethachlor ESA SURR
Precursor Mass
Product Mass
CE
314
314
320
274
328
256
206
356
320
80
80
121
121
121
121
134
80
121
31
27
25
22
26
21
13
31
25
Table 3. Optimized MS transitions for the compounds analyzed.
Results and Discussion
An example chromatogram for the lowest calibration
level (Cal 05, Table 1) is shown in Figure 1. Two of the
compounds, Acetochlor ESA and Alachlor ESA elute very
closely together and are not baseline separated. Therefore,
instead of using peak area to construct the calibration
curve, peak height was used. Additionally, Dimethachlor,
the surrogate compound for the experiment exhibited
peak splitting. Both of the peak’s areas were summed
together for quantitation.
All of the compounds in this analysis were eluted in 22
minutes. Due to the clean nature of the samples, the
chromatographic run time could be shortened to less than
40 minutes, including the time required to equilibrate the
column after the gradient. Furthermore, the Fast-HPLC
capability of the Accela pump could be utilized along with
a 1.9 µm particle size column to reduce the run time even
more, while still providing adequate separation.
Excellent sensitivity was achieved for these analytes,
even without any offline sample preconcentration.
The surrogate and internal standard compounds were
used to illustrate the precision of the method. The %RSD
for the surrogate compound, Dimethachlor ESA, was
2.0% for 25 injections, while Butachlor ESA’s %RSD was
2.3%. Excellent linearity for all of the compounds was
observed. The lowest R2 factor was 0.9972 (Alachlor
ESA), and the highest was 0.9999 (Metolachlor ESA).
Conclusion
This application note demonstrates the
analysis of chloroacetanilide and other
acetamide herbicide degradates using LCMS/MS with no offline sample preparation.
Significant time savings were achieved by
bypassing offline solid phase extraction and
preconcentration. There is the potential for
even higher sensitivity and time savings in
this method if the Thermo Scientific EQuan
automated solution for environmental and
drinking water analysis is used.
Figure 1: Chromatogram of the compounds at the
lowest calibration level (Cal 05). The
concentrations are given in Table 1.
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